The present invention relates to the novel GABAB receptor subtypes GABAB-R1c and GABAB-R2 as well as to a novel, functional GABAB receptor which comprises a heterodimer of GABAB-R1 and GABAB-R2 receptor subunits. The present invention also relates to variants of the receptors, nucleotide sequences encoding the receptors and variants thereof and novel vectors, stable cell lines, antibodies, screening methods, methods of treatment and methods of receptor production.
GABA (xcex3-amino-butyric acid) is the main inhibitory neurotransmitter in the central nervous system (CNS) activating two distinct families of receptors; the ionotropic GABAA and GABAC receptors for fast synaptic transmissions, and the metabotropic GABAB receptors governing a slower synaptic transmission. GABAB receptors are members of the superfamily of 7-transmembrane G protein-coupled receptors. Activation results in signal transduction through a variety of pathways mediated principally via members of the Gi/Go family of pertussis toxin-sensitive G proteins. GABAB receptors have been shown to inhibit N, P/Q and T-type Ca2+ channels in a pertussis toxin-sensitive manner (Kobrinsky et al., 1993; Menon-Johansson et al., 1993; Harayama et al., 1998) and indeed there is also some evidence for direct interactions between GABAB receptors and Ca2+ channels since Ca2+ channel ligands can modify the binding of GABAB agonists (Ohmori et al., 1990). GABAB receptor-mediated Ca2+ channel inhibition is the principle mechanism for presynaptic inhibition of neurotransmitter release. Post-synaptically the major effect of GABAB receptor activation is to open potassium channels, to generate post-synaptic inhibitory potentials. Autoradiographic studies show that GABAB receptors are abundant and heterogeneously distributed throughout the CNS, with particularly high levels in the molecular layer of the cerebellum, interpeduncular nucleus, frontal cortex, olfactory nuclei and thalamic nuclei. GABAB receptors are also widespread in the globus pallidus, temporal cortex, raphe magnus and spinal cord (Bowery et al., 1987). GABAB receptors are an important therapeutic target in the CNS for conditions such as spasticity, epilepsy, Alzheimer""s disease, pain, affective disorders and feeding. GABAB receptors are also present in the peripheral nervous system, both on sensory nerves and on parasympathetic nerves. Their ability to modulate these nerves gives them potential as targets in disorders of the lung, GI tract and bladder (Kerr and Ong, 1995; 1996; Malcangio and Bowery, 1995).
Despite the widespread abundance of GABAB receptors, considerable evidence from neurochemical, electrophysiological and behavioural studies suggests that multiple subtypes of GABAB receptors exist. This heterogeneity of GABAB receptors may allow the development of selective ligands, able to target specific aspects of GABAB receptor function. This would lead to the development of drugs with improved selectivity profiles relative to current compounds (such as baclofen) which are relatively non-selective and show a variety of undesirable behavioural actions such as sedation and respiratory depression. Multiple receptor subtypes are best classified by the differing profiles of agonist and antagonist ligands.
To date screening for GABAB ligands and subsequent structure/activity determinations has relied on radioligand binding assays to rat brain membranes. Further analysis of such ligands in animal models has indicated differences in their behavioural profile. However, due to the absence of cloned GABAB receptors the molecular basis for such differences has not been defined, and therefore it has not been possible to optimise GABAB ligands for therapeutic use.
GABAB receptors were first described nearly 20 years ago (Hill and Bowery, 1981), but despite extensive efforts using conventional expression cloning strategies, for example in Xenopus oocytes, or cloning based on sequence homology, the molecular nature of the GABAB receptor remained elusive. The development of a high affinity antagonist for the receptor finally allowed Kaupmann et al, (1997) to expression clone the receptor from a rat cerebral cortex cDNA using a radioligand binding assay. Two splice variants of the receptor were identified, GABAB-R1a encoding a 960 amino acid protein and GABAB-R1b, encoding an 844 amino acid protein, differing only in the lengths of their N-termini. These two splice variants have distinct spatial distributions within the brain, but both reside within neuronal rather than glial cells. Pharmacologically, the two splice variants are similar, showing binding affinities for a range of antagonists, but about 10 fold lower than those of native receptors, as well as agonist displacement constants which are about 100-150 a fold lower than those of native receptors. These observations have led to speculation that the cloned receptor was a low affinity receptor and an additional high affinity, pharmacologically distinct GABAB receptor subtype could exist in the brain. Alternatively, it was argued that G-protein coupling was inefficient or the receptor was desensitising in the recombinant systems used.
A number of groups working in the area have, however, found that the cloned receptor fails to behave as a functional GABAB receptor either in mammalian cells or in Xenopus oocytes. The present invention describes the cloning of a novel human GABAB receptor subtype, GABAB-R2, the identification of a novel splice variant GABAB-R1c, and the surprising observation that GABAB-R1 and GABAB-R2 strongly interact via their C-termini to form heterodimers. Co-expression of GABAB-R1 and GABAB-R2 allows trafficking of GABAB-R1 to the cell surface and results in a high affinity functional GABAB receptor in both mammalian cells and Xenopus oocytes.
These surpising findings provide a unique opportunity to define GABAB subtypes at the molecular level, which in turn will lead to the identification of novel subtype-specific drugs.
According to one embodiment of the present invention there is provided an isolated GABAB-R2 receptor protein or a variant thereof.
According to another embodiment of the invention there is provided an isolated GABAB-R2 receptor protein having amino acid sequence provided in FIG. 1B, or a variant thereof.
According to a further embodiment of the invention there is provided a nucleotide sequence encoding a GABAB-R2 receptor or a variant thereof, or a nucleotide sequence which is complementary thereto.
According to a further embodiment of the invention there is provided a nucleotide sequence encoding a GABAB-R2 receptor, as shown in FIG. 1A, or a variant thereof, or a nucleotide sequence which is complementary thereto.
According to a further embodiment of the invention there is provided an expression vector comprising a nucleotide sequence as referred to above which is capable of expressing a GABAB-R2 receptor protein or a variant thereof.
According to a still further embodiment of the invention there is provided a stable cell line comprising a vector as referred to above.
According to another embodiment of the invention there is provided an antibody specific for a GABAB-R2 receptor protein or a variant thereof.
According to another embodiment of the invention there is provided an isolated GABAB-R1c receptor protein or a variant thereof.
According to another embodiment of the invention there is provided an isolated GABAB-R1c receptor protein having amino acid sequence provided in FIG. 2, or a variant thereof.
According to another embodiment of the invention there is provided a nucleotide sequence encoding a GABAB-R1c receptor protein or a variant thereof, or a nucleotide sequence which is complementary thereto.
According to another embodiment of the invention there is provided an expression vector comprising a nucleotide sequence as referred to above, which is capable of expressing a GABAB-R1c receptor protein or a variant thereof.
According to another embodiment of the invention there is provided a stable cell line comprising a vector as referred to above.
According to a further embodiment of the invention there is provided an antibody specific for a GABAB-R1c receptor protein or a variant thereof.
According to a further embodiment of the invention there is provided a GABAB receptor comprising an heterodimer between a GABAB-R1 receptor protein or a variant thereof and a GABAB-R2 receptor protein or a variant thereof.
According to a further embodiment of the invention there is provided an expression vector comprising a nucleotide sequence encoding for a GABAB-R1 receptor or a variant thereof and a nucleotide sequence encoding for a GABAB-R2 receptor or variant thereof, said vector being capable of expressing both GABAB-R1 and GABAB-R2 receptor proteins or variants thereof.
According to a further embodiment of the invention there is provided a stable cell line comprising a vector as referred to above.
According to a further embodiment of the invention there is provided a stable cell line modified to express both GABAB-R1 and GABAB-R2 receptor proteins or variants thereof.
According to a further embodiment of the invention there is provided a GABAB receptor produced by a stable cell line as referred to above.
According to a further embodiment of the invention there is provided an antibody specific for a GABAB receptor as referred to above.
According to a further embodiment of the invention there is provided a method for identification of a compound which exhibits GABAB receptor modulating activity, comprising contacting a GABAB receptor as referred to above with a test compound and detecting modulating activity or inactivity.
According to a further embodiment of the invention there is provided a compound which modulates GABAB receptor activity, identifiable by a method as referred to above.
According to a further embodiment of the invention there is provided a method of treatment or prophylaxis of a disorder which is responsive to modulation of GABAB receptor activity in a mammal, which comprises administering to said mammal an effective amount of a compound identifiable by the method referred to above.